Labelled compounds and methods for mass spectrometry-based quantification

Abstract

Methods for peptide and/or protein quantification by mass spectrometry using labeled peptides, wherein multiple labels lead to distinct fragments for the labeled peptides and their unlabeled variant, thus facilitating data analysis and enhancing the potential for quantification. Methods for selecting the label and label position are further given, as well as sets of labeled peptides resulting from or for use in the above-mentioned methods. The methods and substances are especially useful for data-independent or multiplexed parallel reaction monitoring proteomics applications involving peptide quantification.

The invention claimed is: 
     
       1. A method for selecting the label and label position of at least one suitable reference peptide for use in a method for the absolute or relative quantitative analysis of at least one of proteins or peptides, in each case with or without post translational modification(s), using a mass spectrometry method comprising:
 a first step where unlabeled proteins from an endogenous mixture are digested and subsequently digestion products thereof selected, 
 a second step where said digestion products are fragmented, and 
 a third step where a combined fragment spectrum is acquired comprising b-ions as well as y-ions of said digestion products, 
 wherein at least one reference peptide with or without post translational modification(s) is added to said mixture before or after digestion or both, is fragmented, acquired, and stored in said combined fragment spectrum comprising also b-ions and y-ions of said digestion products, 
 wherein said at least one reference peptide is added in a known concentration in case of absolute quantification or in always the same concentration in a series of experiments for relative quantitative analysis, 
 wherein said at least one reference peptide is selectively isotopically labeled by having incorporated:
 one isotopically labeled amino acid forming its very C-terminus or being one of the four terminal amino acids at the C-terminus and one isotopically labeled amino acid forming its very N-terminus, or being one of the four terminal amino acids at the N-terminus, and wherein the isotopically labeled amino acids are unmodified naturally occurring proteinogenic amino acids or amino acids carrying a chemically modifying moiety, wherein said unmodified naturally occurring proteinogenic amino acids or amino acids carrying a chemically modifying moiety comprise one or more atoms that are isotopically labeled such that said one or more atoms are present in the amino acid and not in the chemically modifying moiety 
 
 wherein the position of the label at the C-terminus, or within the four terminal amino acids at the C-terminus, or the position of the label at the N-terminus, or within the four terminal amino acids at the N-terminus, or both, is selected using a procedure which takes into account at least one of the availability of the labeled version of the corresponding amino acid at the respective position or the complexity of the incorporation of the labeled version of the corresponding amino acid at the respective position, and wherein the label and label position is selected so as to be optimized with respect to these parameters. 
 
     
     
       2. The method according to  claim 1 , wherein for the determination of the optimally labeled respective peptide for each combinatorically available possibility having one single label in one position at or close to the C-terminus and one single label in one position at or close to the N-terminus a combined score based on at least one of the availability of the respective labeled version of the corresponding amino acids at the respective positions and the complexity of the incorporation of the labeled version of the corresponding amino acid at the respective position is calculated, and the optimally labeled respective peptide is selected as the one having the best combined score. 
     
     
       3. The method according to  claim 1 , wherein said method for the absolute or relative quantitative analysis of proteins and/or peptides with or without post translational modification(s) using a mass spectrometry method is characterized
 in a first step in which unlabeled proteins from an endogenous mixture are digested and subsequently digestion products thereof are selected, 
 in a second step said digestion products are fragmented, and 
 in a third step a combined fragment spectrum is acquired comprising b-ions and y-ions of said digestion products, 
 wherein at least one reference peptide is added to said mixture before and/or after digestion, is fragmented, acquired, and stored in said combined fragment spectrum comprising also b-ions and y-ions of said digestion products, 
 wherein said at least one reference peptide is added in a known concentration in case for absolute quantification or in always the same concentration in a series of experiments for relative quantitative analysis. 
 
     
     
       4. The method according to  claim 1 , wherein said combined fragment spectrum is acquired using a mass isolation window having a full-range mass isolation window, or a width in the range of 2-1000 Thomson. 
     
     
       5. The method according to  claim 1 , wherein said combined fragment spectrum is acquired using a mass isolation window of 5-30 Thomson. 
     
     
       6. The method according to  claim 1 , wherein said post translational modification is one or more selected from the group consisting of phosphorylation, acetylation, methylation, sulfation, hydroxylation, lipidation, ubiquitylation, sumoylation, glycosylation, oxidation, and carbamidomethylation. 
     
     
       7. Method according to  claim 1 , wherein it involves using DIA or mPRM techniques. 
     
     
       8. A reference peptide or set of reference peptides for use in a method for the absolute or relative quantitative analysis of at least one of proteins or peptides, in each case with or without post translational modification(s), using a mass spectrometry method comprising:
 a first step where unlabeled proteins from an endogenous mixture are digested and subsequently digestion products thereof selected, 
 a second step where said digestion products are fragmented, and 
 a third step where a combined fragment spectrum is acquired comprising b-ions as well as y-ions of said digestion products, 
 wherein at least one reference peptide with or without post translational modification(s) is added to said mixture before or after digestion or both, is fragmented, acquired, and stored in said combined fragment spectrum comprising also b-ions and y-ions of said digestion products, 
 wherein the said at least one reference peptide is added in a known concentration in case of absolute quantification or in always the same concentration in a series of experiments for relative quantitative analysis, 
 wherein said at least one reference peptide this is selectively isotopically labeled by having incorporated:
 one isotopically labeled amino acid forming its very C-terminus or being one of the four terminal amino acids at the C-terminus, and additionally 
 one further isotopically labeled amino acid forming its very N-terminus, or being one of the four terminal amino acids at the N-terminus 
 
 or determined using a method according to  claim 1 , 
 wherein said reference peptide, or at least one or a plurality or all of the reference peptides in the set of reference peptides, is selectively isotopically labeled by having incorporated one isotopically labeled amino acid forming its very C-terminus or being one of the four terminal amino acids at the C-terminus and one further isotopically labeled amino acid forming its very N-terminus, or being one of the four terminal amino acids at the N-terminus, and wherein the isotopically labeled amino acids are unmodified naturally occurring proteinogenic amino acids or amino acids carrying a chemically modifying moiety, wherein said unmodified naturally occurring proteinogenic amino acids or amino acids carrying a chemically modifying moiety comprise one or more atoms that are isotopically labeled such that said one or more atoms are present in the amino acid and not in the chemically modifying moiety. 
 
     
     
       9. The reference peptide or set of reference peptides according to  claim 8 , wherein said reference peptide comprises or consists of 5-100 amino acids. 
     
     
       10. The reference peptide or set of reference peptides according to  claim 8 , wherein in said reference peptide, apart from the isotopically labeled amino acid at or close to the C-terminus and the isotopically labeled amino acid at or close to the N-terminus, not more than one additional amino acid is isotopically labeled. 
     
     
       11. The reference peptide or set of reference peptides according to  claim 8 , wherein in said reference peptide, or in all of said reference peptides, one single isotopically labeled amino acid forms its very C-terminus and one further single isotopically labeled amino acid forms its very N-terminus. 
     
     
       12. The reference peptide or set of reference peptides according to  claim 8 , wherein said at least one reference peptide is selectively isotopically labeled by having incorporated:
 one isotopically labeled amino acid forming its very C-terminus or being one of the four terminal amino acids at the C-terminus and one isotopically labeled amino acid forming its very N-terminus, or being one of the four terminal amino acids at the N-terminus, and 
 wherein the isotopically labeled amino acids are unmodified naturally occurring proteinogenic amino acids or amino acids carrying a chemically modifying moiety, wherein said unmodified naturally occurring proteinogenic amino acids or amino acids carrying a chemically modifying moiety comprise one or more atoms that are isotopically labeled such that said one or more atoms are present in the amino acid and not in the chemically modifying moiety. 
 
     
     
       13. The reference peptide or set of reference peptides according to  claim 8 , wherein said method for the absolute or relative quantitative analysis of proteins and/or peptides with or without post translational modification(s) using a mass spectrometry method comprises:
 a first step in which unlabeled proteins from an endogenous mixture are digested and subsequently digestion products thereof are selected, 
 a second step said digestion products are fragmented, and 
 a third step a combined fragment spectrum is acquired comprising b-ions and y-ions of said digestion products, 
 wherein at least one reference peptide is added to said mixture before and/or after digestion, is fragmented, acquired, and stored in said combined fragment spectrum comprising also b-ions and y-ions of said digestion products, 
 wherein the said at least one reference peptide is added in a known concentration in case for absolute quantification or in always the same concentration in a series of experiments for relative quantitative analysis. 
 
     
     
       14. A method of using one or a set of reference peptides according to  claim 8  for the relative or absolute quantification in protein analysis. 
     
     
       15. The reference peptide or set of reference peptides according to  claim 8 , wherein in said reference peptide, apart from the isotopically labeled amino acid at or close to the C-terminus and the isotopically labeled amino acid at or close to the N-terminus, no additional amino acid is isotopically labeled. 
     
     
       16. The method according to  claim 1 , wherein said combined fragment spectrum is acquired using a mass isolation window having a full-range mass isolation window, or a width in the range of 5-100 Thomson. 
     
     
       17. The method according to  claim 1 , wherein said combined fragment spectrum is acquired using a mass isolation window of 10-25 Thomson. 
     
     
       18. The reference peptide or set of reference peptides according to  claim 8 , wherein said reference peptide consists of 7-30 amino acids. 
     
     
       19. The reference peptide or set of reference peptides according to  claim 8 , wherein said reference peptide consists of 10-20 amino acids. 
     
     
       20. The reference peptide or set of reference peptides according to  claim 8 , wherein said reference peptide comprises or consists of 7-30 amino acids. 
     
     
       21. The reference peptide or set of reference peptides according to  claim 8 , wherein said reference peptide comprises or consists of 10-20 amino acids. 
     
     
       22. The method according to  claim 14  for the relative and/or absolute quantification of complex protein mixtures, in proteomic experiments.